Methods and systems for manipulating particles using a fluidized bed

ABSTRACT

The present invention comprises methods and systems for manipulation of media and particles, whether inert materials or biomaterials, such as cells in suspension cell culture. The methods and systems comprise use of an apparatus comprising a rotating chamber wherein the actions of the combined forces of gravity, fluid flow force and centrifugal force form a fluidized bed within the rotating chamber.

FIELD OF THE INVENTION

The present invention is related to methods, and systems that are usedfor transferring and manipulating particles, including biomaterials,such as cells, cellular components, proteins, lipids, carbohydrates andtissues, and formulations including particles using a fluidized bed.

BACKGROUND

Currently, there are hundreds of biotechnology medicines in thedevelopment pipelines of pharmaceutical and biotechnology companies, andthe numbers are predicted to increase in time. To produce suchbiotechnological products, large numbers of prokaryotic and eukaryoticcells are grown in fermentation systems. Prokaryotic cells, for example,bacterial cells, are physically more resilient than eukaryotic cells.Eukaryotic cells, such as mammalian cells, are more fragile and are moreaffected by steps in the culturing process such as centrifugation,pelleting or re-suspension which are necessary manipulations of thecells for biomanufacturing purposes.

Generally, cells are grown in large stainless-steel fermentation vatsunder strictly maintained and regulated conditions. The cells may be theproduct itself, or the cells may produce a product of interest. Witheither goal, the production of cellular based products is a complicatedprocess. The cells are grown in carefully controlled culture conditionswhich include a balance of temperature, oxygen, acidity, and removal ofwaste products or an excreted product of interest. The growth andactivity of the cells can be interfered with by even slightly alteringthe culture conditions, and can be highly inhibited by actions such asremoval of media, isolation of the cells by spinning out the cells andpacking them in a pellet formed by centrifugation, and resuspension of apacked pellet to reintroduce the cells into the culture conditions.

Many known cell culture methods require significant investment incapital and labor. Cell culture facilities cost millions of dollars tobuild and take several years. There are a limited number of existingfacilities that can be used to produce the products that are currentlyproposed. Cell culture is currently used for production of proteins suchas human insulin, vaccine proteins, enzymes for food processing,biodegradable plastics, and laundry detergent enzymes. Such productsinclude, but are not limited to, therapeutic molecules, vaccines, andantibodies that function as diagnostic tools, therapeutic compounds, inprotein-chips or biosensors.

A growing concern in biomanufacturing is recognition of the extent ofmaterials used for production of cellular products, or how green is thetechnology. This concern looks at the type and amount of resourcesrequired to make therapeutic proteins and other cell culture products,and the wastes generated by mammalian cell culture and microbialfermentation processes. Manufacturing such products is relativelyenvironmentally friendly compared with the production of small-moleculedrugs and commodity petroleum-derived chemicals. However, the processesof cell culture use a lot of water, for example, in batch reactors thathold thousands of liters of culture or fermentation broth. Additionally,even more water, along with consumable processing aids, such as tubing,filters and chromatography processes, are used for downstreampurification. Calculations have shown that for biologics, a currentlarge-scale cell culture process to make a kilogram of monoclonalantibody requires more than 7,600 kg of material, divided as 7,000 kg ofwater, 600 kg of inorganic salts and buffers, which end up in theaqueous waste at the end of the process, 8 kg of organic solvents and 4kg of consumables. For microbial fermentation, 15,500 kg of material isneeded for 1 kg of product, with 15,000 kg being water. Using disposableequipment may add to the waste stream, whereas using reusable materialsadds to the water usage.

What is needed are methods and systems that can be used inbiomanufacturing systems and other types of processes that can be usedwith particles. The particles can be living or inert, includingbiomaterials and all types of cells, hardy cells and cells that requiregentle treatment. What is also needed are methods and systems that donot disrupt cellular growth and activity processes during thebiomanufacturing processes, and may aid in the growth and production ofcells. Additionally, it would be beneficial for methods and systems toprovide green technology advances to the biomanufacturing process.Methods and systems for the transfer and manipulation of particles inefficient methods are also needed.

SUMMARY

The methods and systems disclosed herein comprise methods and systemsfor manipulating particles including inert particles and cells orbiomaterials such as cellular components, proteins, carbohydrates,lipids, and tissues. In one embodiment, the methods and systems of thepresent invention may be used in processes where particles are involved,such as biomanufacturing applications, or for use with suspension cellcultures, such as in transfecting cells using transfection methods. Forexample, the methods and systems disclosed herein can be used forperfusion bioreactor processes and for cell concentration in suspensioncell culture methods. The methods and systems disclosed herein may beused in coating applications, cell or biomaterial selection, orisolation and/or purification of selected biomaterials, such as cells.An apparatus of the methods and systems disclosed herein can function asa continuous flow centrifuge to concentrate cells without the need toremove the cells from the current media, does not subject the cells tothe shear forces found in traditional centrifugation, does not pack orpellet the cells, and avoids the trauma of those actions on livingcells. The methods and systems disclosed herein may be used for inert ornonliving particles and biological particles such as cells.

In another embodiment, the methods and system of the present inventioncan be used to transfer cells from one location, such as a bioreactor,to another location, such as a chamber of a disclosed apparatus, withminimal disturbance to the cells. This capability of moving the cellsallows for a change of media or alteration of the environment of thecells without disturbing the cells, so that the growth and activity ofthe cells is unimpeded. If the aim of the method is to quickly changethe environment of the cells or to affect the growth or activity of thecells, the methods and systems disclosed herein provide for a rapidchange to the cells, without having to centrifuge and pellet the cellsto remove the first environment and resuspend the cells in the newenvironment.

In another embodiment, the methods and systems of the present inventioncan be useful in transfection processes, or other processes that aredesigned to affect the cells individually, such as viral infection. Forexample, the efficiency of transfection of cells in suspension can beaided by the methods and systems disclosed herein. In another example,cells may undergo electroporation techniques. The cells, or a particularsubset of the cells, can be removed from the bioreactor container to achamber of an apparatus and there be affected, such as by transfectionor infection techniques. The cells may be affected by particularchemicals, stimulants, inhibitors, or other factors that alter thecells' activities or growth.

In another embodiment, the methods and systems of the present inventioncan be used to separate cellular subpopulations from a mixed cellularpopulation. For example, cells can be separated based on affinity,density or size. The methods and systems disclosed herein may be used toseparate cellular components or biomaterials including, but not limitedto, proteins, carbohydrates or lipids. For example, a mixture ofproteins can enter a rotating chamber of an apparatus and mediaconditions can be such that a selected population of proteinsprecipitates. Such precipitates form a fluidized bed in the rotatingchamber and are contained within the chamber, and may be removed bychanging the rotation conditions and/or the media flow force.Nonprecipitated proteins are not contained in the fluidized bed and flowthrough the rotating chamber. In one aspect, the methods and systemsdisclosed herein may be used to provide scaffolding materials to cellsor to remove cells from scaffolds, such as cells associated with tissue.In another aspect, the methods and systems disclosed herein can be usedto selectively remove subpopulations of cells from a stationary orgrowing population of cells. In still another aspect, the methods andsystems disclosed herein can conserve media resources.

The methods and systems of the present invention comprise an apparatuscomprising a rotor that rotates about an axis.

According to some embodiments of the present invention, a method formanipulating cells using a fluidized bed includes: rotating a chamberincluding an inlet and an outlet about a substantially horizontal axisto create a centrifugal force field; flowing a first stream containing afirst media and cells into the chamber through the inlet, whereinflowing the first stream acts to create a force which opposes thecentrifugal force; forming a fluidized bed of cells in the chamber,wherein the forces substantially immobilize the cells in the fluidizedbed by the summation of vector forces acting on the cells; collectingthe first media substantially without cells passing through the outletof the chamber; then manipulating the cells in the fluidized bed,wherein said manipulating is selected from the group consisting ofremoving, concentrating, diluting, exchanging media, harvesting,transferring, dispensing, transfecting, electroporating, separating,isolating, extracting, selecting, purifying, coating, binding,physically modifying, and altering the environment; and thereafterremoving the cells from the fluidized bed. Removing the cells from thefluidized bed includes: flowing a second stream into the chamber throughthe outlet, wherein flowing the second stream acts to create a force atleast partially in the same direction as the centrifugal force field;and collecting the cells passing through the inlet of the chamber.

In some further embodiments, the method includes: providing the firststream from a cell culture system prior to flowing the first stream intothe chamber, providing perfusion cell culture conditions to thefluidized bed of cells; exchanging the media; and delivering the cellsand exchanged media to the cell culture system after removing the cellsfrom the fluidized bed.

In some embodiments, manipulating the cells includes transfecting,wherein transfecting includes circulating a transfection streamcontaining a transfection reagent complex through the fluidized bed ofcells one or more times.

In some embodiments, manipulating the cells includes electroporating,wherein electroporating includes: applying an electric current to thefluidized bed of cells; and altering the permeability of the cells. Insome other embodiments, electroporating further includes: flowing acharged molecule stream containing charged molecules into the chamberthrough the inlet before, concurrently with, and/or after applying theelectric current; and incorporating the charged molecules into thecells.

According to some embodiments of the present invention, a method formanipulating particles using a fluidized bed includes: rotating achamber including an inlet and an outlet about a substantiallyhorizontal axis to create a centrifugal force field; flowing a firststream containing a first media and particles into the chamber throughthe inlet, wherein flowing the first stream acts to create a force whichopposes the centrifugal force; forming a fluidized bed of particles inthe chamber, wherein the forces substantially immobilize the particlesin the fluidized bed by the summation of vector forces acting on theparticles; collecting the first media substantially without particlespassing through the outlet of the chamber; then manipulating theparticles in the fluidized bed, wherein said manipulating is selectedfrom the group consisting of removing, concentrating, diluting,exchanging media, harvesting, transferring, dispensing, separating,isolating, extracting, selecting, purifying, coating, binding,physically modifying, and altering the environment; and thereafterremoving the particles from the fluidized bed. Removing the particlescomprises from the fluidized bed includes: flowing a second stream intothe chamber through the outlet, wherein flowing the second stream actsto create a force at least partially in the same direction as thecentrifugal force field; and collecting the particles passing throughthe inlet of the chamber.

In some embodiments, manipulating the particles includes concentratingthe particles, wherein concentrating the particles comprises receivingthe particles in a concentrated particles harvest container afterremoving the particles.

In some embodiments, manipulating the particles includes exchanging themedia, wherein exchanging the media includes: flowing a new media streamcomprising a second media into the chamber through the inlet; andreplacing at least some of the first media in the fluidized bed with thesecond media.

In some embodiments, manipulating the particles includes harvesting,wherein harvesting includes receiving the particles in a particleharvest container after removing the particles.

In some embodiments, manipulating the particles includes dispensing,wherein dispensing includes receiving a measured amount of particles inone or more dispensed cell containers after removing the particles.

In some other embodiments, the particles include a mixed population ofparticles, wherein manipulating the particles includes separating,wherein separating comprises: removing at least some of the particlesfrom the fluidized bed; and collecting the at least some of theparticles passing through the outlet of the chamber. In some furtherembodiments, removing at least some of the particles includes alteringthe centrifugal force field and/or the force of the first stream. Instill further embodiments, the particles are separated by size, density,and/or shape.

In some embodiments, manipulating the particles includes coating theparticles, wherein coating the particles includes: flowing a coatingstream containing a coating material into the chamber through the inlet;and coating the particles retained in the fluidized bed with the coatingmaterial.

According to some embodiments of the present invention, a method forseparating a mixed population of particles includes: rotating a chamberincluding an inlet and an outlet about a substantially horizontal axis;substantially immobilizing an affinity matrix in the chamber; flowing afirst stream containing a first media and a mixed population ofparticles comprising target particles and non-target particles into thechamber through the inlet; retaining target particles in the affinitymatrix in the chamber; and collecting the first media and non-targetparticles passing through the outlet of the chamber. In some furtherembodiments, the method includes: flowing a second stream containing anelution media into the chamber through the inlet; releasing the targetparticles from the affinity matrix; and collecting the target particlespassing through the outlet of the chamber.

According to some embodiments of the present invention, a method forfractionating biomaterials includes: rotating a chamber including aninlet and an outlet about a substantially horizontal axis to create acentrifugal force field; flowing a first stream containing a first mediaand a mixture of biomaterials into the chamber through the inlet,wherein flowing the first stream acts to create a force which opposesthe centrifugal force; selectively precipitating biomaterials from thefirst stream; forming a fluidized bed of the precipitated biomaterialsin the chamber, wherein the forces substantially immobilize theprecipitated biomaterials in the fluidized bed by the summation ofvector forces acting on the precipitated biomaterials; then collectingthe first media and the non-precipitated biomaterials passing throughthe outlet of the chamber; and thereafter removing the precipitatedbiomaterials from the fluidized bed. Removing the precipitatedbiomaterials from the fluidized bed includes: flowing a second streaminto the chamber through the outlet, wherein flowing the second streamacts to create a force at least partially in the same direction as thecentrifugal force field; and collecting the precipitated biomaterialspassing through the inlet of the chamber. In some further embodiments,the biomaterial is protein.

According to some embodiments of the present invention, a method forassociating particles with scaffolding material includes: rotating achamber including an inlet and an outlet about a substantiallyhorizontal axis to create a centrifugal force field; flowing a firststream containing a first media and scaffolding material into thechamber through the inlet, wherein flowing the first stream acts tocreate a force which opposes the centrifugal force; forming a fluidizedbed of the scaffolding material in the chamber, wherein the forcessubstantially immobilize the scaffolding material in the fluidized bedby the summation of vector forces acting on the scaffolding material;flowing a second stream containing a second media and particles into thechamber through the inlet, and retaining at least some of the particleswith the scaffolding material.

According to some embodiments of the present invention, a method forremoving particles from scaffolding material includes: rotating achamber including an inlet and an outlet about a substantiallyhorizontal axis to create a centrifugal force field; flowing a firststream containing a first media and scaffolding material comprisingparticles into the chamber through the inlet, wherein flowing the firststream acts to create a force which opposes the centrifugal force;forming a fluidized bed of the scaffolding material in the chamber,wherein the forces substantially immobilize the scaffolding material inthe fluidized bed by the summation of vector forces acting on thescaffolding material; flowing a second stream containing a dissociationreagent into the chamber through the inlet; removing particles from thescaffolding material; and collecting the removed particles passingthrough the outlet of the chamber.

According to some embodiments of the present invention, a system formanipulating particles, includes: a chamber rotatable about asubstantially horizontal axis to create a centrifugal force field, thechamber having an inlet and an outlet; a bioreactor containing a firstfluid and particles; at least one pump in fluid communication with therotating chamber and the bioreactor; a fluid manifold in fluidcommunication with the rotating chamber, wherein the manifold includes aplurality of spaced apart valves that are automatically selectivelyclosed and opened during use; a controller in communication with the atleast one pump and the valves, wherein the controller directs: (i) thevalves to open and close, (ii) the flow rates of the at least one pump,(iii) the rotational speed of the rotating chamber, and (iv) a flowvelocity of a first stream containing the first fluid and particles fromthe first fluid source into the chamber through the inlet, wherein inoperation, the flow velocity of the first stream from the first fluidsource into the chamber through the inlet acts to create a force whichopposes the centrifugal force, thereby forming a fluidized bed ofparticles in the chamber, wherein the forces substantially immobilizethe particles in the fluidized bed by the summation of vector forcesacting on the particles. In some further embodiments, the controllerfurther directs a flow velocity of a second stream containing a secondfluid from a second fluid source into the chamber through the outlet,wherein in operation, the flow velocity of the second stream from thesecond fluid source into the chamber through the outlet acts to create aforce at least partially in the same direction as the centrifugal forcefield, thereby removing the particles from the fluidized bed. In somefurther embodiments, in operation, the particles are collected throughthe inlet of the chamber and returned to the bioreactor after beingremoved from the fluidized bed. In still further embodiments, the atleast one pump includes a bi-directional pump.

According to some embodiments of the present invention, a system formanipulating particles, includes: a rotating chamber comprising spacedapart inlet and outlet ports sized and configured to apply a centrifugalforce and an opposing fluid flow force to particles therein; a primarypump in fluid communication with the rotating chamber and a fluidsource; a fluid manifold in fluid communication with the rotatingchamber, wherein the manifold includes a plurality of spaced apartvalves that are automatically selectively closed and opened during use;a fluid buffer wash source in communication with the manifold; asecondary pump in fluid communication with the fluid buffer wash sourceand the manifold, wherein the secondary pump has active on and offperiods, and wherein in an active on period, the secondary pump has ahigher flow rate than the primary pump, wherein the secondary pumpresides proximate the fluid buffer wash source attached to a first armof the manifold, and wherein a second arm of the manifold has opposingfirst and second end portions arranged so that the second arm extendsaround the secondary pump with the first end portion attached above thesecondary pump and the second end portion attached to the first armbelow the secondary pump, and wherein the second arm includes a firstone of the valves; and a controller in communication with the primarypump, the secondary pump and the valves, wherein the controller directs:(i) the opening and closing of the valves, (ii) the flow rates of theprimary and secondary pumps, (iii) the rotational speed of the rotatingchamber to create a gravitational force of between about 25-15,000×g,(iv) an average flow velocity of the fluid from the fluid source throughthe chamber at between about 20-300 mm/min, wherein in operation, thesystem has a wash cycle that flushes defined segments of the manifoldwith buffer from the buffer wash source after initial loading of therotating chamber with target media to thereby cleanse dead legs in themanifold.

In some further embodiments, the manifold includes a first flow paththat extends between the rotating chamber and a bioreactor, the inputpath having a bioreactor valve positioned proximate the bioreactor, andthe second arm of the manifold merges into the input flow path upstreamof the bioreactor valve and includes two serially spaced apart valvestherebetween, such that, in the buffer wash cycle, the two seriallyspaced apart valves and the bioreactor valve are open when the secondarypump is on and the first valve in the second arm is closed, so that adead leg at the bioreactor valve is flushed. In some furtherembodiments, the manifold includes a second flow path that extendsbetween the rotating chamber and the second arm of the manifold with awaste line extending to a waste container upstream of the second arm ofthe manifold with a waste valve in the waste line, the second flow pathincluding a second flow path valve that resides upstream of the secondarm of the manifold, wherein, in the buffer wash cycle, after the deadleg at the bioreactor is flushed, the bioreactor valve is closed, andthe second flow path valve is open to flush a dead leg associated withthe secondary flow path valve. In still further embodiments, the systemincludes a third arm of the manifold in communication with thebioreactor, the third arm of the manifold including a valve

DESCRIPTION OF FIGURES

FIG. 1 illustrates forces involved in the present invention.

FIG. 2 is an illustration of the mathematics governing the motion of aparticle due to the effect of gravity on that particle when it isrestrained in a centrifugal field that is opposed by liquid flow.

FIG. 3 is an illustration of the resultant motion of a particle underthe constraints of FIG. 2.

FIG. 4 is a mathematical evaluation of the immobilization of conditionsat a given radius.

FIG. 5 is an analysis of the balance of centrifugal forces and flowvelocity forces in a rotating cylindrical chamber.

FIG. 6 is an analysis of the balance of centrifugal forces and flowvelocity forces in a rotating conical chamber.

FIG. 7 is an illustration of a three-dimensional array of particles in arotating conical chamber.

FIG. 8 is an illustration of the inter-stratum buffer regions in athree-dimensional array of particles in a rotating conical chamber.

FIG. 9 is a mathematical analysis of the intra-stratum flow velocityvariation in a two-dimensional array of particles in a rotating conicalchamber.

FIG. 10 is an illustration of an example of a conical-shaped chamber andthe boundary conditions which determine those dimensions.

FIG. 11 is an analysis of the positional variation of the centrifugaland flow velocity forces in the chamber of FIG. 10 at a flow rate of 10mL/min.

FIG. 12 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 13 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 14 is a graph showing the viable cell density when using anexemplary method and system of the present invention.

FIG. 15 is a graph showing the viable cells using an exemplary methodand system of the present invention.

FIG. 16 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 17 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 18 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 19 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 20 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 21 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 22 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 23 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 24 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 25 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 26 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 27 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 28 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 29 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 30 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 31 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 32 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 33 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 34 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 35 is a schematic diagram of an exemplary method and system of thepresent invention.

FIG. 36 is a schematic diagram of an exemplary method and system of thepresent invention.

DETAILED DESCRIPTION

The present invention now is described more fully hereinafter withreference to the accompanying drawings, in which some embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Like numbers refer to like elements throughout. In the figures, thethickness of certain lines, layers, components, elements or features maybe exaggerated for clarity.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, steps, operations, elements, components, and/or groupsthereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andrelevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

It will be understood that when an element is referred to as being “on”,“attached” to, “connected” to, “coupled” with, “contacting”, etc.,another element, it can be directly on, attached to, connected to,coupled with or contacting the other element or intervening elements mayalso be present. In contrast, when an element is referred to as being,for example, “directly on”, “directly attached” to, “directly connected”to, “directly coupled” with or “directly contacting” another element,there are no intervening elements present. It will also be appreciatedby those of skill in the art that references to a structure or featurethat is disposed “adjacent” another feature may have portions thatoverlap or underlie the adjacent feature.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of “over” and “under”. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

As used herein, the term “particles” includes inert and livingmaterials, and includes, but is not limited to cells, cellularorganelles, enzymes, biomolecules such as proteins, lipids,carbohydrates, inert materials such as polymeric or copolymericmaterials that are nano or microparticles and other types of nano ormicroparticles.

As used herein, the term “cell culture system” refers to any system orapparatus in which cells are grown, including, without limitation,mammalian, avian, insect, fungal, and bacterial cells. In oneembodiment, a cell culture system refers to a system in which cells aregrown in suspension.

As used herein, the term “substantially without particles” refers to anamount of particles that is less than 20% of the total amount ofparticles in the chamber, e.g., less than 15, 10, 5, or 1%.

As used herein, the term “substantially horizontal” refers to an axisthat is within about 20 degrees of horizontal, e.g., within about 15,10, 5, or 1 degree of horizontal.

As used herein, the term “substantially immobilized” means that theparticles may move to a small extent within the chamber but do not exitthe chamber.

As used herein, the term “fluid” includes liquids and gases.

As used herein, the term “biomaterials” refers to materials that arepart of a cell or other living structure, e.g., proteins, peptides,nucleic acids, lipids, carbohydrates, membranes, organelles, etc.

As used herein, the term “physically modifying” refers to the physicalalteration of a particle (e.g., cell), e.g., a change in physical and/orchemical structure, covalent binding to another molecule, incorporationof a molecule within the particle, etc.

As used herein, the term “altering the environment” refers to a changein the milieu surrounding the particle, e.g., a change in media,addition of one or more compounds to the media, a change in theconcentration of a compound within the media, etc.

The methods and systems disclosed herein comprise methods and systemsfor the manipulation of particles, such as inert particles or livingparticles, such as cells in cell culture, using a fluidized bed. Usefulapplications of the methods and systems include, but are not limited to,movement of particles (e.g., cells, either prokaryotic or eukaryotic)from one location to another, concentrating or diluting of particles(e.g., cells), such as increasing or decreasing the number of cells/mL,changing of media conditions, performing actions on the particles (e.g.,cells) or changing the environment of the particles (e.g., cells), suchas transfecting the cells or providing specific chemical activators orinhibitors to the cells, and providing a controlled measured dispensingof particles or cells into other vessels, such as into vials or othercontainers.

The methods and systems of the present invention may comprise anapparatus comprising a rotor that rotates in a plane substantiallycoaxial with the gravitational axis. The apparatus may be outfitted withcomponents to allow for the flow of liquid media. The apparatussubstantially immobilizes the particles that form a fluidized bed by useof the summation of the vector forces acting on each particle.Embodiments of such apparatus have been disclosed in U.S. Pat. Nos.5,622,819; 5,821,116; 6,133,019; 6,214,617; 6,660,509; 6,703,217;6,916,652; 6,942,804; 7,347,943; and U.S. patent application Ser. Nos.12/055,159 and 11/178,556, each of which is incorporated by reference inits entirety. Though cells and particles are light in weight, their massis non-zero. Consequently, gravity has a significant effect on thesuspended particle or cell, and this effect will increase with time. Theweight of the suspended particles or cells causes these particles tosettle to the lowest regions of the container, disrupting the balance offorces which initially suspended them in the chamber. As is seen inprior art devices, particles tend to aggregate and the aggregation ofthese particles into a larger particle results in an increasedcentrifugal effect which causes the aggregates to migrate to longerradii, eventually causing destabilization of the fluidized bed.

An apparatus used in the methods of the present invention take advantageof the relationships inherent in (1) Stoke's Law and the theory ofcounterflow centrifugation; (2) the geometrical relationships of flowvelocity and centrifugal field strength; and, (3) the effect ofhydraulic pressure on media and particles. The methods of the presentinvention comprise apparatus that are capable of forming a fluidized bedof particles by the immobilization of three-dimensional arrays ofparticles such as cells, by employing rotation around a horizontal axisand balancing forces including gravity, centrifugal force from therotation and a liquid flow force provided by the media stream enteringthe chamber or container holding the particles.

The theoretical basis of the apparatus of the present invention utilizesa novel method to immobilize suspended particles. A proper applicationof Stoke's Law, in combination with provision for the effect of gravity,which acts on the immobilized suspended particles, results in amathematical relationship which allows for the relative immobilizationof such particles. The effect of gravity can be compensated for by thechoice of rotational axis as is shown in FIG. 1. If rotation about thehorizontal axis (y) is chosen instead of rotation about the verticalaxis (z), as is most common in biological centrifugation apparatus andmethods, then the effect of gravity on immobilized particles will belimited to action solely in the x-z plane. Since this is the same planein which both the centrifugal as well as the liquid flow related forcesare constrained to act, the motion of a suspended particle at any pointin a rotational cycle is the resultant of the sum of the three types offorces acting upon it. Rotation about the horizontal axis means that thesuspended particle is rotating substantially coaxial with thegravitational force axis.

As is shown in Inset A of FIG. 2, where the plane of the Figure is thex-z plane, the effect of gravity (Fg) on the position of a particlesuspended in a radially-directed centrifugal field (Fc) while an exactlyequal and opposing force supplied by an inwardly-directed flowing liquid(Fb) is directed toward the particle, can be calculated by theevaluation of equations 1-4 where (k) represents the downwarddisplacement in the x-z plane imparted by gravitational forces during anangular rotation of the rotor position equal to (a). Analysis of themotion of a particle under these constraints and for [2πx(k/a)]<R (a lowmass particle) results in the determination that the motion is periodic;that is, the particle motion results in a return to its starting placeafter a complete rotation of 360 degrees (after equilibrium is reached).As is shown in FIG. 2, the effect of gravity on the motion of a particlewhich is otherwise immobile as a result of the opposing equality of thecentrifugal and flow-related forces results in a decrease in radialposition in quadrants I and II, and an exactly equal radial lengtheningin quadrants III and IV. Thus, the radial distance of the particle fromthe axis of rotation also exhibits a periodic motion over the course ofa full rotation of 360 degrees. It should be noted that, mathematically,measurement of the periodicity of motion requires only one rotation ifmeasurement begins at either 90 or 180 degrees whereas two fullrotations are required if measurement begins at either zero or 180degrees, since a new equilibrium radial distance different from theoriginal results in the latter case.

The effective motion of a particle through a complete rotational cycleis shown in the inset of FIG. 3. If the sides of a container in whichthe particle is suspended are labeled 1 and 2, then the motion of theparticle over the course of one rotational cycle would describe a circlewith its center displaced toward the “leading edge” side of theparticle's container. Thus, a particle suspended in a centrifugal fieldwhich is opposed by an equal liquid flow field will be constrained toperiodic motion (and thus is effectively immobilized) if the balance ofthe radially-directed forces can be maintained over the course of itsmovement.

A graphical representation is shown in FIG. 4, in which the axis ofrotation is now the (y) axis. Under these conditions the hypothesis ofSanderson and Bird can now be restated and applied to immobilization ofparticles. There is a radial distance along the z axis (rz) which, whenevaluated by Eqn. 3, represents a position in which the particle isrelatively immobilized in a centrifugal field which is exactly opposedby an inwardly-directed liquid flow, even in the presence of agravitational field. Furthermore, a simplification of Stoke's Law(Eqn. 1) under the conditions of uniform particle size, shape, anddensity and a homogeneous liquid flow results in Eqn. 2, where it isobvious that the Sedimentation Velocity of a particle (SV) is a simplelinear function of the applied centrifugal field. Similarly, Eqn. 3 canthen be rewritten under the same conditions to yield Eqn. 4, whereliquid Velocity (V in Eqn. 3) has been replaced by liquid Flow Velocity(FV). Equation 4 suggests that there is a continuum of liquid flowvelocities and applied centrifugal fields which could be matched by theevaluation of constant (C), all of which would satisfy the requirementof relative particle immobilization. Further, if the liquid flowvelocity could be varied as a function of (z), there could be a separateapplication of this equation at each radial distance. Consideration ofthe implications of Eqn. 4 is important for the relative immobilizationof three-dimensional arrays of particles as opposed to theimmobilization of two-dimensional arrays of particles at a single radialdistance from the rotational axis.

If the chamber in which a particle is located is cylindrical (as isgraphically depicted in FIG. 5) and if a liquid is flowed into thischamber from the end of the chamber most distal to the axis of rotation,then it is obvious that the flow velocity of this liquid flow (asdefined in Eqn. 1, FIG. 5) will have a single value at all points notoccupied by layers of particles. As a consequence, if a two-dimensionalarray of particles is in positional equilibrium at a particular radialdistance (A1), as is indicated in Eqn. 2, (where CF is the centrifugalfield strength and FV is the liquid flow velocity) then particles forcedto occupy positions at radial distances either greater than or smallerthan A1, such as those located in FIG. 5 at A2 or A3, will necessarilybe presented with an inequality of restraining forces which will resultin net translation of the particles. Thus, those particles located atA2, a longer radial distance than A1, will experience a greatercentrifugal force than those at A1 and will necessarily migrate tolonger radial distances (Eqn. 3). Conversely, particles initiallylocated at A3 would experience a reduced centrifugal field and wouldmigrate to shorter radial distances (Eqn. 4). Thus, it is believed thatit is not possible to form a three-dimensional array of particles in aparallel-walled chamber such as that of FIG. 5.

If, however, the chamber has a geometry such that its cross-sectionalarea increases as the rotational radius decreases, as is graphicallydisplayed in FIG. 6, then it is possible to form three-dimensionalarrays of immobilized particles, for example, cells. This is aconsequence of the fact that the microscopic flow velocity of the liquidflow varies inversely as the cross-sectional area (Eqn. 1) while therelative centrifugal field varies directly as the rotational radius(Eqn. 2). Thus, if values of flow velocity and rotation velocity arechosen such that a two-dimensional array of particles is immobilized atrotational radius A1 (Eqn. 3), then it is possible to adjust the “aspectratio” of the side walls of the chamber such that those particlesinitially located at radial distance A2 could also experience either ansimilar equality of forces or, as is shown in Eqn. 4, an inequality offorces which results in net motion back toward the center of thechamber. A similar argument may be applied to particles located at A3(see Eqn. 5). Although the geometry of the chamber as depicted in FIG. 6is that of a truncated cone, note that other geometries could bealternatively used—subject to the constraint that the cross-sectionalarea of the chamber increases as the rotational radius decreases. Thus,as is depicted in FIG. 7, it is possible to construct athree-dimensional array of particles in a varying centrifugal fieldopposed by a liquid flow field if the chamber geometry chosen allows fora flow velocity decrease greater than or equal to the centrifugal fieldstrength decrease as the rotational radius decreases. In the geometrychosen in FIG. 7, that of a truncated cone, the two-dimensional arraysof particles at each rotational radius (Rc) will each be constrained tomotion toward that radius where the opposing forces are exactly equal.

While, at first glance, the description presented above would suggestthat the net effect of the mismatch of forces at all radii other thanthat which provides immobilization would result in a “cramming” of allparticles into a narrow zone centered on the appropriate radius, such isnot the case. As is shown graphically in FIG. 8, as each layer ofparticles approaches an adjacent layer, it will move into a region wherea “cushioning effect” will keep each layer apart (the horizontal arrowsin FIG. 8). The explanation for the inability of adjacent layers ofparticles to interdigitate is a consequence of an analysis of themicroscopic flow velocity profile through each layer. In FIG. 9, asingle representative stratum of spherical particles confined to aparticular radial distance in a chamber layer of circular cross-sectionis presented. The ratio of the diameters of the particles to thediameter of the cross-section of FIG. 9 is 12:1. While the magnitude ofthe flow velocity of the liquid through unoccupied portions of thechamber cross-section can be quantified simply from the chamberdimensions at that point, the flow velocity through a region occupied bya stratum of particles will necessarily be much greater than that in theabsence of a stratum of particles because of the greatly reducedcross-sectional area through which the liquid must travel. As is shownin the graph in FIG. 9, the increase in flow velocity through a stratumof the above dimensions is more than double that determined in the freespace just adjacent to the stratum on each side. This microscopicincrease in local flow velocity in the region of each stratumeffectively provides a “cushion” which keeps each adjacent stratumseparate, and when cells are the particles, a fluidized bed of cellsresults.

For example, in the case of a chamber geometry of a truncated cone, itis preferable that the most distal region of the truncated cone be theregion where an exact equality of centrifugal forces and liquid flowvelocity is achieved. The “aspect ratio” (the ratio of the small radiusof the truncated cone to the large radius of the truncated cone) of thetruncated cone is determined by the simultaneous solution of the twoequations presented in FIG. 10. In Eqn. 2, the desired boundarycondition of immobility for that “lowest” stratum of particles ispresented. It states that the intrinsic sedimentation rate of theparticle due to gravity (SR) times the relative centrifugal fieldapplied at that radial distance (RCF) be exactly equal to the magnitudeof the liquid flow velocity (FV) at that point. In Eqn. 1, a desiredboundary condition at the opposite surface of the array of particles ispresented. When the methods require the retention of particles withinthe container or chamber, a boundary condition wherein the product of SRand RCF is twice the magnitude of the flow velocity at that radialdistance is chosen. Simultaneous solution of the desired boundarycondition equations is used to solve for the ratio of the conic sectiondiameters when the upper diameter and conic length is known. Where themethods require the expulsion or removal of particles from thecontainer, the forces are altered and not balanced, while rotationcontinues. For example, the liquid force may not balanced by thecentrifugal force, or the liquid force and the centrifugal force may acttogether in the same direction, and the particles exit the chamber.

FIG. 11 is a profile of the relative magnitudes of the flow-relatedforces and the centrifugal forces across a chamber of conicalcross-section which has dimensions in this example of: largediameter=6.0 cm, small diameter=3.67 cm and depth=3.0 cm. The RelativeSedimentation Rate is defined as the product of the intrinsicsedimentation rate of a particle due to gravity in a media at itsoptimal temperature and the applied centrifugal field. For a given flowrate (in this example 10 mL/min) into a chamber of the indicateddimensions, where the proximal end of the biocatalyst immobilizationchamber is 9.0 cm from the rotational axis, the product of the intrinsicparticle sedimentation rate due to gravity and the angular velocity is aconstant at the given flow rate in order to satisfy the desired boundaryconditions (see FIG. 10). In other words, the angular velocity need notbe specified here since its value depends only on the particularparticle type to be immobilized. The dotted line in FIG. 11 displays thelinear variation in the centrifugal field strength from the bottom tothe top of the biocatalyst immobilization chamber, while the solid linedisplays the corresponding value of the flow velocity. At the bottom ofthe chamber (the most distal portion of the chamber), the forces areequal and a particle at this position would experience no net force. Atthe top of the chamber, a particle would experience a flow-related forcewhich is only one-half of the magnitude of the centrifugal field andwould thus be unlikely to exit the chamber, even in the presence of anearby region of decreasing cross-sectional area (the chamber liquidexit port), where flow velocities will increase markedly.

It should be clear from the foregoing that, subject to the necessarycondition that the cross-sectional area increases as rotational radiusdecreases, there are other geometrical chamber configurations whoseshape could be manipulated in order to establish boundary andintermediate relationships between the applied centrifugal field and theliquid flow velocity forces at any radial distance in order to establishdesired resultant force relationships in the three-dimensional particlearrays. In practice, however, it is undesirable to utilize geometrieswith rectangular cross-sections as a result of the anomalous effects ofcoriolis forces which act in a plane transverse to the rotational plane.In the case of rectangular cross-sections, these otherwise unimportantforces can contribute to interlayer particle motion.

The effect of gravitational forces acting on the individual particlemasses which acts independently of the applied centrifugal forces areeven less important than was indicated earlier. In particular, since thebasic effect of gravity on an otherwise immobilized particle is toeither cause radial lengthening or radial shortening, such a motion of aparticle will necessarily bring it either into a region of increasedflow velocity magnitude (longer radii) or decreased flow velocitymagnitude (shorter radii) with only a much smaller change in centrifugalfield strength. As a consequence, the periodic motion of a particle dueto gravitational effects on its intrinsic mass will be severely dampenedin the presence of such unbalanced opposing force fields and will amountto, in the case of low mass particles, a vibration in place.

In some embodiments of the invention, the rotor is rotated at a speedsufficient to create a centrifugal force of about 25 to about 15,000×g,e.g., about 50 to about 5000×g, e.g., about 75 to about 500×g, e.g.,about 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000,13,000, 14,000, or 15,000×g or more or any subrange therein, dependingon the type of particle in the chamber. For example, a suitablecentrifugal force for mammalian cells can be in the range of about 25 toabout 1000×g, whereas a suitable centrifugal for lighter particles(e.g., bacteria or biomaterials (protein, DNA)) can be in the range ofabout 5000 to about 15,000×g. In certain embodiments, the average fluidflow velocity through the chamber (measured at ⅓ chamber height from thetip of the chamber) is in the range of about 5 to about 800 mm/min,e.g., about 20 to about 300 mm/min, e.g., about 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, or 800 or more or any subrange therein, depending on thetype of particle in the chamber. In other embodiments, the density ofthe fluidized bed can be in the range of about 0.1×10⁸ to about 5.0×10⁸,e.g., about 0.5×10⁸ to about 2.0×10⁸, e.g., about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or5.0×10⁸ or more or any subrange therein, depending on the type ofparticle in the chamber.

The methods disclosed herein comprise use of an apparatus thatsubstantially immobilizes the particles to form a fluidized bed ofparticles by use of the summation of the vector forces acting on eachparticle. Embodiments of such an apparatus have been disclosed in U.S.Pat. Nos. 5,622,819; 5,821,116; 6,133,019; 6,214,617; 6,334,842;6,514,189; 6,660,509; 6,703,217; 6,916,652; 6,942,804; 7,029,430;7,347,943; and U.S. patent application Ser. Nos. 11/384,524; 12/055,159and 11/178,556, each of which is incorporated by reference in itsentirety.

In one aspect, this apparatus can comprise a cylindrical rotor bodymounted on a motor-driven rotating shaft. The rotor body can be fixed inposition on the rotating shaft by means of locking collars, and issupported on either side of the rotor by bearings. In another aspect,bioreactor chambers can be mounted on the rotor, and liquid flows can beintroduced into and removed from the bioreactor chambers by means ofliquid channels within the rotating shaft.

In some embodiments of the invention, part are all of the fluid pathwithin the apparatus and/or into and out of the apparatus is composed ofdisposable materials. The use of a completely disposable fluid path, aswell as a closed system operational design, permits compliance withcurrent good manufacturing practice (cGMP).

The apparatus and the chambers therein can be any size suitable for themethods of the invention. Depending on the size of the apparatus, therotor body can contain one or more chambers, e.g., 1, 2, 3, 4, 5, 6, 7,8, or more chambers. The total volume of all of the chambers in therotor body can range from about 0.5 mL or less to about 5 L or more,e.g., 10, 15, or 20 L or more. In some embodiments, the total chambervolume is about 0.5, 1, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500,600, 700, 800, or 900 mL, or about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5L. For large scale bioprocessing applications, the total chamber volumecan be, for example, in the range of about 250 mL to about 1 L or more.For small scale processes (e.g., research laboratory use, clinicallaboratory use, blood processing, etc.), the total chamber volume maybe, for example, in the range of about 0.5 mL to about 100 mL or less.

When the rotor body contains more than one chamber, in some embodiments,each chamber can have its own separate fluid path. In other embodiments,multiple chambers can be connected in serial or parallel fluid pathways.In certain embodiments, different processes in the methods of theinvention can be carried out in different chambers in a single rotorbody.

In one aspect, the methods disclosed herein comprise the use of anapparatus that is capable of forming a fluidized bed of particles byemploying rotation around an axis. In another aspect, the methods andsystems of the current invention can be used where the introduction of,or the generation of, gases within a liquid medium in the chamber isdesired. In other embodiments of the methods and systems disclosedherein, the presence or absence of gas in solution or out of solution inthe liquid medium is immaterial to the methods and systems. Thus, thehydraulic pressure of the liquid-containing parts of the system,including the chambers and liquid lines leading to and from thechambers, may or may not be maintained at a hydraulic pressuresufficient to fully dissolve the necessary quantity of input gas and toinsure the solubility of any produced gases.

Previous bioprocessing systems have been directed towards manufacturingof protein therapeutics while the cells are discarded. In contrast, thepresent invention provides methods for gently manipulating cells withimproved recovery and decreased contamination by intracellular proteinsfrom damaged cells. The present methods impart low shear and minimalpressure drops on the cells and provide clog-free and continuousoperation compared to current cell retention systems, such ascentrifugation-based systems, filtration-based systems, sedimentationsystems, ultrasonic systems, and hydrocyclone systems. The presentinvention further provides an integrated system for processing of cellsand other particles that reduces the number of processing steps as wellas processing time.

The methods of the present invention can be used with any type of cellculture system (e.g., bioreactors, flasks, dishes, or other growthchambers), including perfusion culture, batch culture and fed-batchculture systems. The methods of the present invention also can be usedwith any type of cells, including, without limitation, bacteria, yeast,plant cells, insect cells, avian cells, mammalian cells, human cells,cells lines, primary cells, embryonic or adult stem cells, etc. Themethods of the invention can also be carried out with fluids thatcomprise cells (e.g., bodily fluids such as blood, urine, saliva,cerebrospinal fluid, etc.) as well as other sources of cells (e.g.,cells cultured on microparticles, tissue samples (e.g., biopsies oraspirates), samples of cultured primary cells (e.g., stem cells,allogeneic cells), etc.).

An aspect of the methods and systems of the present invention comprisesproviding perfusion cell culture conditions to cells. For example,batch, fed-batch, and perfusion bioreactor processes are widely used inthe manufacturing of biotherapeutics. In comparison to batch andfed-batch processes, perfusion bioreactor processes lead to higher celldensities, titers and product quality as the product and toxicby-products are continually removed while nutrients are constantlyreplenished. Most perfusion processes generally run for much longerduration and require smaller equipment than batch or fed-batchprocesses. Although the perfusion process has several advantages overthe traditional batch and fed-batch process, one of the major hurdles inperfusion process is retention of cells throughout the process. Mostcommonly, cells are retained in the bioreactor by using either acentrifugation or filtration based device. Centrifugation based devices,wherein the rotation is around a vertical axis, which means wherein therotation is substantially transverse to the gravitational axis, canproduce shear stress and nutrient deprivation to the cells in forming apellet and these conditions lead to low viability of the cellpopulation. Filtration based devices can suffer from clogging issuesrelated to the filters and produce shear stress on cells, which caninhibit cell growth and activity.

In one aspect, when a stream of media containing cells passes into theapparatus comprising a chamber for rotating cells, a fluidized bed ofthe cells is formed with a continuous perfusion of media through it. Thecells are then in an environment of minimal shear and one which providesa constant supply of oxygen and nutrients to the cells. For example,cells may be removed from a stationary bioreactor container andtransferred to an apparatus comprising a rotating chamber while themedia is transmitted to and through the chamber. A fluidized bed ofcells can be formed within the chamber that is being rotated at a rateto retain the cells in relation to the fluid force of the media.

One aspect is shown in FIG. 12 and is an example of perfusion bioreactormethods and systems of the present invention. Cells are located in thebioreactor 1 and are growing in the media provided. A media stream isinitiated by providing media from a media container 10 using a pump 11through pathway 12, which is generally tubing. Media and cells flow outof bioreactor 1, via pathway 2 and through a pump, such as abi-directional pump 3, to an apparatus 4 comprising a rotating chamber5. As the media and cells flow into the rotating chamber 5, the cellsare retained in the rotating chamber 5, and the media flows out of theapparatus 4 via pathway 6. The media follows pathway 6 through valve 7,and via pathway 8, to a container 9 for spent media, or harvested media,or may be discarded (pathway not shown).

At a desired timepoint or condition, such as when the rotating chamber 5is almost full, the cells can be transferred to another location, suchas being returned to bioreactor 1. In one aspect, as the rotatingchamber continues to rotate, the fluid force can be changed by reversingthe flow direction, and with the centrifugal force and the liquid forceacting at least partially in the same direction, all or a portion of thecells may leave the rotating chamber.

As an example of this method and system, see FIG. 13. Media is pumpedvia pathway 14 and can be supplied from media supply container 10, orfrom another source of media. Media may also be provided from thebioreactor 1 (pathway not shown). Valve 13 is opened and media flows viapathway 6 into an apparatus 4 comprising a rotating chamber 5. Thecells, which were retained within the rotating chamber 5, flow from therotating chamber 5 via pathway 2 through a pump 3, such as abi-directional pump, and into bioreactor 1. The media may be pumpedthrough the rotating chamber 5 and the pathways, or tubing, for adesired amount of time. The cells previously retained in the rotatingchamber 5 return to the bioreactor, and are mixed in the population ofcells. After a desired amount of time, the flow direction is reversedagain, valve 13 is closed, valve 7 is opened, and the perfusion cycle,as shown in FIG. 12 and FIG. 13 is repeated. Using this perfusion cycle,the bioreactor cells are provided with fresh media continuously and thespent media is removed.

It is to be understood in the exemplary methods and systems disclosedherein, such as in the Figures, that the methods and systems disclosedherein are not limited to only the containers, pathways or pumps asshown. For example, those skilled in the art can readily substitute abi-directional pump with one or more pumps, and pathways are intended toprovide fluid flow conduits, such as provided by tubing or piping.

Example 1 discloses a comparison of using the methods and systems of thecurrent invention to create a perfusion bioreactor process by providingfresh media and removing spent media from the bioreactor, meanwhilecapturing cells leaving the bioreactor in the spent media in an rotatingchamber, and returning those captured cells to the bioreactor withlittle or no interference with the growth or activity of the cells. Theperfusion cycle of fluid flow in one direction away from the bioreactorin which spent media is removed, followed by a reversal of the fluidflow so that captured cells and media return to the bioreactor may berepeated during the bioreactor run. The perfusion cycle may be repeatedone or more times, for example, two times, 3 times, 4 times 5 times, 6times, 7 times, 8 times, or in a range of 1-25 times, 1-50 times, 1-100times, 1-300 times, 1-400 times, 1-500 times, 1-1000 times per batchperiod, or per day, or per week, or per month, depending on the needs ofthe cells, which can be determined by someone skilled in the art. Thedirection of the flow of the media which creates a fluid force in therotating chamber, may be reversed in a method or system of the presentinvention every 0.5 minute, 1 minute, 2 minutes, 3 minutes, 4 minutes,every 5 minutes, every 10 minutes, every 15 minutes, every 20 minutes,every 30 minutes, every 40 minutes, every 45 minutes, every 50 minutes,every 60 minutes, every 2 hours, every 3 hours, every 4 hours, every 5hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every10 hours, every 11 hours, every 12 hours, from every 0.5 minutes toevery 24 hours and any range in between.

An alternative media flow can be utilized in a perfusion bioreactor.Looking at FIG. 12, fresh media is fed constantly into the bioreactorfrom media supply 10 via pathway 12 by pump 11. Cells and spent mediaare pumped out of the bioreactor 1, and cells are captured in therotating chamber 5, and spent media is removed by pathway 6, thoughvalve 7 into container 9. In the other half of the perfusion cycle, butnot shown in FIG. 13, the media flow is reversed and media may beprovided by having pathway 14 originate in the bioreactor 1 or othersource, such as media supply from 10 (not shown in diagram). The mediais pumped from the bioreactor, through valve 13, and through therotating chamber 5 of apparatus 4, and along pathway 2 by pump 3 andback into the bioreactor via pathway 2. Any cells that might beentrained with the media coming from the bioreactor are either washed onthrough the rotating chamber 5 because the centrifugal force and thefluid flow force are at least partially aligned, or when the fluid flowis reversed again in the start of a new cycle, any cells present arecaptured in the rotating chamber 5 and form a fluidized bed of cells.

The methods and systems of the present invention can be used with anytype of cell culture system (e.g., bioreactor) bioreactor for at leastthe methods and systems disclosed herein. In one aspect, the methods andsystems can be used with any size bioreactor, plastic, glass orstainless steel bioreactors, and can be used with stationary or portablebioreactors. In another aspect, the methods and systems allow forbioreactors wherein the cell viability is very high because there is areduction in the stresses on cells. In still another aspect, the methodsand systems disclosed herein can be used with and attach to a cellculture system.

A method and system of the present invention comprises use of anapparatus comprising a rotating chamber as a continuous centrifuge. SeeFIG. 16. In one aspect, cells can be located in the bioreactor 1. Mediaand cells flow out of bioreactor 1, via pathway 2, through valve 15 andthrough a pump, such as a bi-directional pump 3, to an apparatus 4comprising a rotating chamber 5. Valve 17 is closed. The cells areretained in the rotating chamber 5, and the media flows out of apparatus4 via pathway 6, and as shown to a container 16 for clarified media. Themedia may be recycled or may be discarded (pathway not shown). The cellsform a fluidized bed in the rotating chamber 5.

As the rotating chamber continues to rotate, the fluid force is changedby reversing the fluid flow direction, and with the centrifugal forceand the liquid force acting at least partially in the same direction,all or a portion of the cells may leave the rotating chamber.

As an example of this method and system, see FIG. 17. Media is pumpedfrom a media container, such as container 16, via pathway 6 to theapparatus 4 and the rotating chamber 5. The cells leave the rotatingchamber 5 via pathway 2, through bi-directional pump 3. Valve 17 isopened, valve 15 is closed, and the media and cells flow via pathway 19and into the container 18. In one aspect, the chamber 5 of the apparatus4 does not need to stop rotating throughout this process. The cycle ofpumping cells and media from one container, containing the cells withinthe rotating chamber and removing the cells from the rotating chamber toa different container can be repeated multiple times, as disclosedabove, for example, to concentrate cells from large volumes.

Another method and system of the present invention comprises use of anapparatus comprising a rotating chamber, media and/or buffer exchangeduring cell culture or harvest. See FIG. 18. For example, media exchangecan be used to provide fresh media or to switch cells to a differentmedia, e.g., growth media, storage media, dispensing media, transfectionmedia, etc. Media exchange can be used to remove contaminants from thecell culture, e.g., to remove small particulate impurities (such asparticles of plastic generated from the disposable tubing and/orchamber), to remove intracellular proteins or cell debris from damagedor lysed cells, to remove free virus or other biological contaminants,etc. In some embodiments, the media exchange is at least 90% effectivein replacing old media with new media (at least 90% of the old media isremoved), e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%effective. In other embodiments, effective media exchange can beaccomplished with minimal use of new media, e.g., less than about 10chamber volumes, e.g., less than about 9, 8, 7, 6, 5, 4, 3, or 2 chambervolumes. In further embodiments, media exchange can be carried out withhigh retention of cells, e.g., at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or moreretention. For example, cells are located in the bioreactor 1. Media andcells flow out of bioreactor 1, via pathway 2, through valve 15 andthrough a pump, such as a bi-directional pump 3, to an apparatus 4comprising a rotating chamber 5. Valves 17 and 21 are closed. The cellsare retained in the rotating chamber 5, and the media flows out ofapparatus 4 via pathway 6, and as shown to a container 16 for clarifiedmedia. The cells form a fluidized bed in the rotating chamber 5.

An example of adding new media or buffer is shown in FIG. 19. As therotating chamber continues to rotate, valve 21 is opened and a new mediaor buffer is pumped via pathway 20 through a pump, which may bebi-directional pump 3 or a unidirectional pump (not shown), to apparatus4. The cells in the rotating chamber 5 are exposed to and surrounded bythe new media or buffer, and the new media or buffer may completely orpartially replace the original media or buffer. The cells remain in therotating chamber 5, and the new media/buffer leaves apparatus 4 throughpathway 6 to another container, such as container 16.

Once the new media or buffer is at a desired concentration, such asreplacing 100% of the original buffer or media, the cells may bereturned to the bioreactor and continue to grow in the presence of thenew media or buffer. In one aspect, for example, the rotating chambercan continue to rotate, and the fluid force is changed by reversing thefluid flow direction. In another aspect, with the centrifugal force andthe liquid force acting at least partially in the same direction, all ora portion of the cells may leave the rotating chamber.

In this aspect, media is pumped from a media container, such ascontainer 16, via pathway 6 to the apparatus 4 and the rotating chamber5. The cells leave the rotating chamber 5 via pathway 2, throughbi-directional pump 3. Valve 17 is closed, valve 15 is open, and mediaflows via pathway 2 into the bioreactor. The cells, which were containedwithin the rotating chamber 5, flow from the rotating chamber 5 viapathway 2 through the bi-directional pump 3 and into the bioreactor 1.

Alternatively, as shown in FIG. 20, the cells can be harvested. Cellharvesting can be used, for example, to recover cells that have beenexpanded in culture for different purposes, e.g., cells that have beenproduced for use as a vaccine, cell samples from a subject (e.g.,allogeneic cells or embryonic or adult stem cells that have beenexpanded for readministration to the subject), etc. Media is pumped froma media container, such as container 16, via pathway 6 to the apparatus4 and the rotating chamber 5. The cells leave rotating chamber 5 viapathway 2, through bi-directional pump 3. Valve 17 is open, valve 15 isclosed, and media flows via pathway 19 into the container 18. The cells,which were contained within the rotating chamber 5, flow from therotating chamber 5 via pathway 2 through the bi-directional pump 3 andinto the container 18. In one aspect, the rotating chamber of theapparatus 4 does not need to stop rotating throughout this process. Thecycle of pumping cells and media from one container, containing thecells within the rotating chamber and removing the cells from therotating chamber to a different container can be repeated multipletimes, as disclosed above. This can, for example and according to oneaspect, provide new media or new buffers to the cells at any time duringthe growth and/or activity of the cells, or to provide a media or bufferfor harvesting or storage. After the cells are washed with the media orbuffer, the rotating chamber is emptied of cells by reversing the fluidflow. Media/buffer exchange applications as disclosed herein may be usedprior to transfection, cell dispensing, seeding a bioreactor, or anyother steps in the maintenance, growth, harvesting or treating of cellsin culture.

FIGS. 21-23 show an aspect of the methods and systems disclosed hereinwherein the methods and systems can be used as a cell dispenser to fillvials with cells. In this application, cells are concentrated, a mediaand/or buffer exchange may occur, and then cells are transferred fromthe rotating chamber by reversing the fluid flow of media, or new mediaand/or buffer, to dispense the cells in vials or bottles, or any desiredcontainers. The cell dispenser can be used to generate cell banks, fillvials for cell therapy, for freezing, for dispensing into well plates,or any container for which a measured amount of cells is desired.

In the example shown in FIG. 21, cells are located in the bioreactor 1.Media and cells flow out of bioreactor 1, via pathway 2, through valve15 and through a pump, such as a bi-directional pump 3, to an apparatus4 comprising a rotating chamber 5. Valves 17 and 21 are closed. Thecells are retained in the rotating chamber 5, and the media flows out ofapparatus 4 via pathway 6 and 25, and through valve 24 to a container 23for waste. The cells form a fluidized bed in the rotating chamber 5.

As illustrated in FIG. 22, the media and/or buffer is exchanged whilethe cells are in the rotating chamber 5. New media and/or buffer ispumped from the new media and/or buffer container 22 through valve 21via pathway 20 to a pump, such as bi-directional pump 3, and intoapparatus 4 comprising a rotating chamber 5. The new media and/or bufferflows through and out rotating chamber 5, through pathway 6 and 25 tovalve 24 and into a container 23.

FIG. 23 illustrates an example of moving the cells from a rotatingchamber to a dispensing container. In one aspect, the cells have formeda fluidized bed within the rotating chamber 5. The direction of thefluid force is changed so that the fluid force and the centrifugal forceare at least partially aligned in the same direction. In another aspect,this change can occur by using a bi-directional pump 3 to reverse thefluid flow. New media and/or buffer is provided from container 26 viapathway 28 and valve 27 to pathway 6 and into rotating chamber 5 inapparatus 4. The cells and media leave rotating chamber 5 via pathway 2,through pump 3, through valve 17, through pathway 19 and into adispensing container 45.

During the loading of cells into the rotating chamber, stagnant areas influid pathways may be contaminated with, for example, culture media.These stagnant areas, or “dead legs,” may occur near valves and may berinsed with clean media/buffer to get a complete buffer wash. As usedherein, “clean” media/buffer may mean that the media/buffer is sterileor substantially sterile. FIG. 36 illustrates exemplary methods andsystems for achieving this result. A main pump P1 controls the flow ofmedia through the chamber. A secondary pump P2 is set to run at a higherrate than the primary pump P1. During the loading of cells, dead legsmay occur at or near valves V1 and V7. To rinse the dead legs, after thecells are loaded and while the buffer rinsing is occurring the secondarypump P2 may be turned on and valve V8 closed. Valves V3 and V5 arealready open due to buffer washing. Valve V1 is opened for a short timeto flush the dead leg at that valve (with the excess flow from pump P2).Pump P2 is then turned off, and valve V8 is opened and valve V1 isclosed for the remainder of the buffer wash time. Before commencing theharvest routine, pump P2 is again turned on, and valve V8 is closed.Valve V7 is then opened for a short time to flush the adjacent dead leg.Pump P2 is then turned off and valve V7 is closed and valve V8 isopened. At this point an uncontaminated harvest may occur. In someembodiments, bubble detectors BS1, BS2, BS3, and BS4 sense the end of afluid stream, and can therefore trigger the next stage of the process(e.g., washing). It is noted that bubble detectors or other flowdetectors may be employed in any of the methods and systems of thepresent invention.

Cells in culture can be acted on to provide new products, aid in thegrowth of the cells, or alter the original activity of the cells. Forexample, cells may undergo transfection or infection procedures thatintroduce DNA or RNA into the cells. The materials introduced inside thecells may be DNA or other nucleic acids or constructs, proteins,chemicals, carbohydrates, vaccines or viral particles, or otheractivities that are known for affecting cells.

Transfection is routinely used, for example, to introduce genes into atarget cell. In comparison to adherent cultures, suspension culturesgenerally exhibit lower transfection efficiency. This may be due toreduced contact time between the transfection reagent complex and thecells. The methods and systems of the present invention may be used fortransfection. In one aspect, the target cells may be exposed to thenucleic acids of interest (DNA and/or RNA) along with the correctbuffers or other compounds that make up a transfection reagent complex.Any transfection technique known in the art that is suitable for use inthe apparatus of the present invention can be used, including, withoutlimitation, calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, electroporation,DNA-loaded liposomes, lipofectamine-DNA complexes, and viral-mediatedtransfection/infection. In one embodiment, the transfection reagentcomplex can be a viral vector (e.g., a viral particle) containing anucleic acid of interest, e.g., retrovirus, lentivirus, adeno-associatedvirus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus,Epstein-Barr virus, or adenovirus vector. The cells may be exposed tothe transfection reagent complex by providing the transfection reagentcomplex to a fluidized bed of cells present in a rotating chamber of anapparatus. The cells may be grown in shake flasks or bioreactors. FIG.24 and FIG. 25 show an example of this method and system. Exposing thecells in a higher density found in a rotating chamber increases contacttime between the transfection reagent complex and cells. As thetransfection reagent complex traverses through the interstitial spacebetween the cells, contact time is increased. As shown in FIG. 24, cellsare located in bioreactor 1. Media and cells flow out of bioreactor 1,via pathway 2, through valve 15 and through a pump, such as abi-directional pump 3, to an apparatus 4 comprising a rotating chamber5. The cells are retained in the rotating chamber 5, and the media flowsout of apparatus 4 via pathway 6, through pathway 25, valve 24 and intocontainer 23. The cells form a fluidized bed in the rotating chamber 5.

FIG. 25 shows a transfection method and system, according to one aspect,though this method and system may be used for infection or any otheractions upon the cells, or provision of particular compounds or factorsto the cells. In this aspect, the method and system are not limited bythe material being provided to the cells. For example, in a transfectionprocedure, the transfection reaction complex comprising at least DNAand/or RNA or a nucleic acid construct of interest, is contained incontainer 29 and is pumped out of container 29 via pathway 30 throughvalve 31 and pathway 32 by a pump, such as bi-directional pump 3 to anapparatus 4 comprising a rotating chamber 5 containing a fluidized bedof cells. The transfection reaction complex flows through the fluidizedbed of cells in rotating chamber 5, through pathway 6 and 33 to valve34, through pathway 35 and into container 29 so that the transfectionreaction complex recirculates. The transfection reaction complex may berecirculated as long as needed to ensure adequate exposure of the cells.

In one aspect, after the transfection reaction complex has been presentwith the cells for an adequate amount of time, for example for 1-60minutes, or for 1-3 hours, or for the desired time of exposure, therecycling of the transfection reaction complex fluid stream is reversed.See FIG. 26. In this aspect, the transfection reaction complex fluidflows from container 29, through pathway 35, through valve 34, throughpathways 33 and 6 into the rotating chamber 5 of apparatus 4. This fluidflow change may be controlled by a pump, such as a bi-directional pump3. With this reversal of fluid flow, the fluid force and the centrifugalforce are acting at least partially in the same direction and the cellsare removed from the rotating chamber 5. The cells and media pass fromthe rotating chamber 5, through pump 3, via pathway 2 to valve 15 andinto a container, such as bioreactor 1 or shaker flasks. In one aspect,the methods and systems disclosed herein can easily be used to performlarge scale transfections using a smaller amount of transfection complexthan would be required otherwise. Alternatively, and in another aspect,should a media exchange be required prior to transfection, a simplemedia exchange step, as shown above, can be added prior to transfection.With the increased contact time between the cells and the transfectionmaterial, improved transfection efficiency can be found.

Another aspect of the current invention comprises methods and systemsfor affecting cells or biomaterials, for example, by use ofelectroporation techniques. For example, an electric current can alterthe permeability of cell membranes, and allow for the entry of nucleicacids or other charged molecules into the cell or cellular component,such as mitochondria. FIG. 29 illustrates one aspect of such methods andsystems. In this aspect, media containing cells is pumped from acontainer 1 of cells in media, via pathway 2, via a bidirectional pump 3into an apparatus 4 comprising a rotating chamber 5. The cells withinthe rotating chamber 5 are acted on by an electric current field, andthe permeability of the cellular membrane is altered. In variousaspects, one or more pulses of an electric field may be applied to thecells to change the permeability of the membrane, or to affect the cellsin some manner. The media surrounding the cells may comprise one or morenucleic acid sequences, proteins, salts or ions that may cross the cellmembrane with altered permeability. After electroporation, the cells mayreturn to container 1 from rotating chamber 5 via pathway 2 through apump, such as a bi-directional pump 3, and into container 1. Theelectroporated cells may be stored in an alternative container 8, bypumping the media and cells from the rotating chamber 5 via pathway 2through a pump, such as a bi-directional pump 3, through valve 7 andinto container 8. The media may be pumped through the rotating chamber 5and the pathways, or tubing, for a desired amount of time. Waste mediaand/or cells may be removed from the rotating chamber 5 via pathway 6 towaste container 9.

FIG. 30 shows another aspect of electroporation methods. Mediacontaining cells is pumped from a container 1 of cells in media, viapathway 2, via a bidirectional pump 3 into an apparatus 4 comprising arotating chamber 5. The cells are retained within the rotating chamber5, and are concentrated in the chamber as a fluidized cell bed. Thecells may be washed at this point and fluid that contains molecules(stored in container 10) can be re-circulated through the fluidized cellbed via pathways 11 and 12. An electric field is applied in shortpulse(s) to incorporate the molecules into the cells. Once theelectroporation is complete, the cells can be collected for furtherprocessing. The cells may be pumped to one or more sites.

In general, electroporation methods comprise exposing particles,including biomaterials, such as cells or cellular components withmembranes, to an electric field of appropriate strength to alter thepermeability of the particle or the cell membrane. Charged molecules,such as nucleic acids, DNA, RNA, charged ions, proteins, enter theparticles more easily because of the altered permeability. As anexample, cells are concentrated in a rotating chamber of an apparatuscomprising a rotating chamber where the cells form a fluidized bed inthe chamber. The cells may be washed, and a media containing chargedmolecules can be added to the fluidized bed. In certain embodiments, thecells may be exposed to the charged molecules before, concurrently,with, and/or after the electric field is applied. An electric field isapplied, for example, in short pulses, and the cellular membranes arealtered. The type, strength, and length of the electric pulse can beoptimized for each cell type. For mammalian cells, in certainembodiments, the pulse is in the form of a square wave or anexponential. The voltage of the pulse can be in the range of about 50 toabout 1000 V, e.g., about 100 to about 500 V, e.g., about 50, 100, 200,300, 400, 500, 600, 700, 800, 900, or 1000 V or more or any subrangetherein. The length of the pulse can be in the range of about 1 ms toabout 100 ms, e.g., about 5 to about 50 ms, e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 ms or more or any subrange therein. The cells can bepulsed more than once, e.g., 2, 3, 4, or 5 times or more. Chargedmolecules or ions enter the cells. The cells may then be transferred toanother holding tank or returned to the general population, usingreverse flow, and the process can be repeated.

The methods and systems disclosed herein can be used to separate apopulation of cells, including but not limited to, separating based ondensity and/or size. In one embodiment, fluid containing differentpopulations of cells, such as cells that differ in size or in density,can be fed into a rotating chamber. Examples of fluids containingdifferent populations of cells include, without limitation, bodilyfluids (such as blood, urine, saliva, cerebrospinal fluid, etc.),digested tissue samples, co-cultures of different cell types, etc. Inthe rotating chamber, the cells can be separated by modulating the fluidflow (fluid force) and/or centrifugal force. A fluidized bed of cellscan be formed in the rotating chamber. By changing the rate of rotation,thus altering the centrifugal force applied to the cells, by changingthe fluid flow, thus altering the fluid force on the cells, or bychanging both the rate of rotation and the fluid flow, particular cellsor a subpopulation of cells that have a similar size and/or density canbe separated from the fluidized bed and removed from the rotatingchamber. Once the fluid force and the centrifugal force are adjustedappropriately, lighter and/or smaller cells can exit out of the rotatingchamber in the media stream. See FIG. 27. After a cell bed is formed,fresh media or buffer could be used to separate another subpopulation ofsimilar cells by once again adjusting the fluid force and/or thecentrifugal force. This process can be repeated several times toseparate multiple subpopulations of cells that differ by density and/orsize. Finally, heavier/larger cells are harvested by reversing the flowof fresh media (FIG. 28).

In FIG. 27, for example, cells comprising a mixed population of largeand small cells are located in container 1. Media and cells flow out ofcontainer 1, via pathway 2, through valve 15 and through a pump, such asa bi-directional pump 3, to an apparatus 4 comprising a rotating chamber5. The cells are retained in rotating chamber 5, and the media flows outof apparatus 4 via pathway 6, through valve 36 and into container 37. Toremove smaller and/or lighter cells, the fluid force is altered byincreasing or decreasing the rate media is pumped into rotating chamber5, and/or the centrifugal force is increased or decreased by alteringthe rate of rotation of the rotating chamber 5. Either one of the fluidforce or centrifugal force, or both can be changed to remove asubpopulation of cells. The remaining cells reform a fluidized bed, andthen either one or both of the fluid force or centrifugal forces arechanged to remove another subpopulation of similar cells, as describedabove, and may be repeated as often as desired. When the procedure forremoving subpopulations of cells is completed, and the desiredpopulation remains within the rotating chamber, the fluid flow isreversed so that the fluid force and the centrifugal force are at leastpartially, if not completely, aligned. The cells can then be removedfrom the rotating chamber to another container. In methods of cellculture, one may want to select small cells or large cells, for example,and the present invention provides methods and systems that can providethe segregation of cells to select for the desired type.

One aspect of a method and system for harvesting heavier/larger cells isillustrated in FIG. 28. In this aspect, the fluid flow is reversed, andmedia is provided from container 10 via pathway 43 to valve 42 and viapathway 44 to apparatus 4 comprising a rotating chamber 5. The cells andmedia flow out of rotating chamber 5 via pathway 2 to a pump, such asbi-directional pump 3, via pathway 41 to valve 39, and into container 38via pathway 40.

The methods and systems disclosed herein may be used for selection,purification, or enrichment of particular cells, biomaterials, orparticles. For example, affinity methods may be used to select for aparticular target, such as a cell. Affinity targets can include specificcell types, e.g., embryonic or adult stem cells, pluripotent cells,tissue specific cells, or cells of a specific differentiation stage. Anaffinity matrix may be contained within a rotating chamber and a mixedpopulation which comprises one or more targets can be transferred intothe rotating chamber. The affinity matrix can be any suitable particle,bead, or resin that binds to an affinity target and is capable offorming a fluidized bed, e.g., standard chromatography material. Theaffinity matrix can comprise a material that binds the affinity target,e.g., antibodies (polyclonal, monoclonal, fragments, Fc regions, etc.),protein A and protein G containing materials, dyes, receptors, ligands,nucleic acids, etc. Cells or particles that have affinity for the matrixwill be bound or retained by the matrix, while other particles or cellsmay exit the chamber. The exiting material may be recirculated so thatit reenters the rotating chamber for access to the affinity matrixagain. The cells or particles bound or associated with the affinitymatrix may be released by adding an elution media in the media stream inthe rotating chamber. The released cells or particles may be collectedafter release from the affinity matrix. See FIG. 31.

Another aspect comprises methods and systems for enrichment of cells orparticles, for example, by use of an affinity matrix. For example, inFIG. 31, systems and methods for separating target cells or particlesare disclosed, according to one aspect. In this aspect, media containingtarget cells is pumped from a container 1 of heterogeneous population ofcells in media, via valve 13, via a bidirectional pump 3, via pathway 2and into an apparatus 4 comprising a rotating chamber 5. The cells,which are retained within the rotating chamber 5, are acted on by theaffinity matrix, and the target cells, +ve, are retained by the affinitymatrix, and the other cells, −ve, flow through the rotating chamber. Themedia and cells exiting the rotating chamber may be recirculated for oneor more passes through the rotating chamber and the affinity matrix, andexiting cells, −ve, pass through valve 8 and are stored in container 11.An elution media is pumped from container 12 through valve 9 via abidirectional pump 3, via pathway 2 into the rotating chamber 5 ofapparatus 4. The eluting media breaks the association between the targetcells and the affinity matrix so that the target cells, +ve, are removedfrom the matrix. The target cells flow out of rotating chamber 5 throughpathway 6, via valve 7 and are contained in container 10.

In another example, cells expressing a specific surface receptor can beisolated from a mixed population of cells. Beads coated with antibodyfor the receptor function as the affinity matrix and can be immobilizedwithin a rotating chamber. As a mixed population of cells is introducedin the system, cells that exhibit the receptor are bound by the antibodyon the matrix and are retained within the rotating chamber, whereascells without the receptor flow through the rotating chamber. In adifferent embodiment, the mixed population of cells can be exposed tothe antibody prior to entering the rotating chamber. Beads coated with amaterial that binds antibodies (e.g., protein A or protein G) can beimmobilized in the chamber. As the mixed population of cells isintroduced in the system, those cells that are bound to the antibodywill bind to the affinity matrix and retained within the chamber. In oneaspect, cells with the receptor may be released from the antibody matrixby flowing a media containing, for example and without limitation, areleasing agent such as trypsin or a soluble molecule recognized by theantibody, through the rotating chamber, and collect the cells releasedby action of the releasing agent.

Another aspect comprises methods and systems for fractionation ofproteins or other biomaterials. See FIG. 32. Media containing a mixtureof proteins is pumped from a container 1, via valve 10, via abidirectional pump 3, and flows via pathway 2 into an apparatus 4comprising a rotating chamber 5. The protein mixture media is capable ofselectively precipitating proteins, and the precipitated proteins areretained within rotating chamber 5. Such conditions are known to thoseskilled in the art, and may include pH, ionic strength, chemicals (e.g.,ammonium sulfate), or protein concentrations. The precipitated proteinsare retained within rotating chamber 5 due to the balanced forces actingon the precipitated proteins. The non-precipitated proteins are notretained within rotating chamber 5, and may flow though pathway 6 tocontainer 8. The precipitated proteins accumulate as a fluidized bed andmay be collected by reversing the pump flow and flow through pathway 2,via a bidirectional pump 3, through valve 7 and into container 9. Theprocess may be repeated and conditions in the media may be altered sothat different proteins are precipitated under each differing condition(a protein fraction), and the individual fractions are stored, each in adifferent container 9.

Another aspect of the methods and systems of the present inventioncomprises associating cells or other biomaterials with scaffolds orremoving cells or other biomaterials from scaffolds. As used herein,scaffold includes three dimensional structures in which, for example,cells can be associated, embedded, whether internally or externally, orboth. Such scaffolds may be natural, such as the natural architecturefound in a tissue comprising cells, or in a tissue in which cells havebeen removed, or may be made from synthetic or natural materials to forma three dimensional shape. For example, a collagen scaffold may be usedby using a native structure such as a decellularized blood vessel, orfrom collagen molecules, used as scaffolding material, forming a randomthree dimensional shape. Other examples of scaffolding material include,but are not limited to alginate and proteoglycan. Another example of themethods and systems disclosed herein is shown in FIG. 33. Mediacontaining scaffolding material is pumped from a container 10, via valve8, via a bidirectional pump 3, via pathway 2 into an apparatus 4comprising a rotating chamber 5. The scaffolding material is retained inrotating chamber 5. The scaffolding material may or may not becross-linked. Media containing cells is pumped from a container 1, viavalve 7, via a bidirectional pump 3, and flows via pathway 2 into therotating chamber 5 of apparatus 4. The cells are retained within therotating chamber and are associated with or embedded on and within thescaffolding material. The scaffolding material may or may not becross-linked. The order of addition of scaffolding material and cellsmay be reversed, with cells entering the rotating chamber 5 first,followed by addition of scaffolding material. The rotating chamber maybe opened and the three dimensional scaffold with associated cells canbe removed.

Another aspect comprises removing cells or other biomaterials from ascaffold. In some embodiments, cells can be removed from samples oftissues. In other embodiments, cells can be removed from artificialsupports on which they have been grown in culture, e.g., microparticlesor other types of scaffolds. For example, see FIG. 34, where smallpieces of tissue comprising cells are pumped from container 1 throughvalve 7 via a bidirectional pump 3, and flows via pathway 2 into anapparatus 4 comprising a rotating chamber 5. The small pieces of tissueare retained within rotating chamber 5 due to the balanced forces actingon the pieces of tissue. A dissociation reagent, such as for example andwithout limitation, trypsin, collagenase, or other digestive enzymes, ispumped from container 10, via valve 8, via a bidirectional pump 3, andflows via pathway 2 into the rotating chamber 5 of apparatus 4. Thedissociation reagent acts on the immobilized pieces of tissue, and cellsare removed from the tissue and flow out of the rotating chamber throughpathway 6 to container 9. The released cells may enter a second rotatingchamber (not shown) to be washed or placed in a different media. In thisaspect, there is minimal exposure of the cells to dissociation reagentsor harsh conditions, which results in less damage to the cells.

The methods and systems of the present invention also comprise methodsand systems for treating biomaterials. For example, and as illustratedin FIG. 35, methods and systems can be used coating of particles, forexample, cells, cellular components or other biomaterials. For example,cells can be encapsulated in a particular material or more than onematerial (e.g., in different layers). As shown in FIG. 35, mediacontaining particles is pumped from a container 1, via valve 10, via abidirectional pump 3, and flows via pathway 2 into an apparatus 4comprising a rotating chamber 5. The particles can be retained withinrotating chamber 5 due to the balanced forces acting on the particles. Acoating material, provided in a fluid medium, which may be a liquid orgas, is pumped from a container 12, through valve 9 via a bidirectionalpump 3, and flows via pathway 2 into the rotating chamber 5 of theapparatus 4 to coat the particles. Excess coating material may flowthough pathway 6 via valve 8 to container 12, and repeat the coatingprocess in one or more cycles, or may flow through valve 7 into a wastecontainer 11. The particles in the fluidized bed are uniformly coated,and may be transferred to the rotating chamber for other actions, suchas drying or storage (not shown). One or more coats may be applied tothe particles, and the coats may comprise the same materials or maycomprise different materials. In one aspect, for example, a first coatcan be one material and a second coat can be a different material.

The methods and systems shown herein may be used to transport cells fromone container to another container, or back to the original containerwithout exposing the cells to centrifugation, filtration, and pelletinghazards. The examples shown herein can be modified for any procedureswherein cellular manipulation, isolation, concentration, media exchangeor easy transfer of cells is desired. Such procedures are contemplatedby the present invention.

As discussed above, the methods and systems of the present invention mayemploy a rotor (which may be driven by a motor), one or more valves,and/or one or more pumps. These components may be controlled by one ormore controller. In other words, a single controller may control all thecomponents or some or all of the components may have dedicatedcontrollers. In some embodiments, the controller(s) direct: 1) theopening and closing of the valve(s), 2) the flow rates of the pump(s),3) the rotational speed of the rotor, either directly or via the motor,4) the rotational speed of the chamber, and/or 5) a flow velocity offluid and/or particles from a fluid source, such as a bioreactor. Insome embodiments, the controller(s) may direct the application of anelectric field, such as, for example, an electric field applied in theelectroporation techniques described in more detail above.

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES Example 1

The methods and systems disclosed herein were used as a perfusionbioreactor process. In this example, the methods and systems as shown inFIG. 12 and FIG. 13 were used to remove spent media from a bioreactorcontainer, and to return media to the bioreactor to perfuse the cellswithin the bioreactor. There were some cells in the media leaving thebioreactor (the spent or used media). The cells and the media wereflowed through a rotating chamber which retained or captured the cellspresent in the spent media, and the spent media continued out of therotating chamber into a container for disposal. After a certain timeperiod, the fluid flow was reversed, and media, such as fresh new media,was flowed into the rotating chamber. The centrifugal force and thefluid flow force within the chamber acted at least partially in the samedirection, and the cells flowed out of the rotating chamber and werereturned to the bioreactor, along with the fresh media. This comprisedone perfusion cycle. After a desired time, the perfusion cycle wasrepeated by reversing the fluid flow and pumping spent media and cellsout of the bioreactor again. A stirred tank bioreactor process isreferred to herein as a batch process.

To compare the perfusion process to a batch process in which cells werenot removed, two 15 L Applikon stir-tank bioreactors containing 5 L ofCDCHO media (Invitrogen) were inoculated with CHO-S cells (Invitrogen)to a cell density of 0.26×10⁶ cells/mL. Cell counts were performed dailyto monitor growth and viability of cultures. Continuous perfusioncycles, in which the spent media was removed from the bioreactorcontainer, along with some cells, wherein most of the cells werecaptured by a rotating chamber, while the fluid flowed in one direction,and then fluid flow was reversed so that media, such as fresh media, andthe captured cells were returned to the bioreactor container, wasinitiated in one of the bioreactors on Day 3.

In the perfusion bioreactor, a perfusion rate was maintained at 5 L/dayby media feed at a rate that matched the rate of the harvest of spentmedia. The 5 L/day rate gave a one volume/day exchange of media for thebioreactor. The cells leaving the bioreactor were captured and returnedto the bioreactor.

The volume of the bioreactor was 5 L, temperature was maintained at 37°C., and the pH was 6.9 to 7.4. The dissolved oxygen was 30%, with animpeller speed of 120 rpm, low air, and a carbon dioxide overlay to aidin pH control. The rotating chamber had a capacity of 30 mL, and wasrotated at 800 rpm. The exchange rate was one volume/day and the cycletime was 30 minutes.

In the perfusion cycle, every 30 minutes, the fluid flow was reversed,so that in the rotating chamber, the centrifugal force and the fluidforce were no longer balanced in opposition to each other, and the fluidforce and the centrifugal force worked in the same direction to removethe fluidized cell bed of captured cells from the rotating chamber, andreturn the cells and media to the bioreactor. After initiation of theperfusion cycle process, viable cell density (VCD) consistentlyincreased in the perfusion process sample in comparison to the batchprocess sample (FIG. 14). The experiment was terminated on Day 8, afterfive days of removal, immobilization and return of the cells, every 30minutes. At Day 8, VCD in the perfusion bioreactor reached 19.2×10⁶cells/mL while VCD in the batch bioreactor was 3.9×10⁶ cells/mL.Viability remained >95% throughout the experiment (FIG. 15), indicatingthat the movement of the media and the capture of the cells was notdeleterious to the cells. The data show that using the systems andmethods disclosed herein for perfusion bioreactor growth of cellsproduces higher cell mass than batch process using traditional stir-tankbioreactor.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. The invention is defined by the following claims, withequivalents of the claims to be included therein.

1. A method for manipulating particles using a fluidized bed, the methodcomprising: rotating a chamber about a substantially horizontal axis tocreate a centrifugal force field, the chamber comprising an inlet and anoutlet; flowing a first stream containing a first media and particlesinto the chamber through the inlet, wherein flowing the first streamacts to create a force which opposes the centrifugal force; forming afluidized bed of particles in the chamber, wherein the forcessubstantially immobilize the particles in the fluidized bed by thesummation of vector forces acting on the particles; collecting the firstmedia substantially without particles passing through the outlet of thechamber; then manipulating the particles in the fluidized bed, whereinsaid manipulating is selected from the group consisting of removing,concentrating, diluting, exchanging media, harvesting, transferring,dispensing, transfecting, electroporating, separating, extracting,isolating, selecting, purifying, coating, binding, physically modifying,and altering the environment; and thereafter removing the particles fromthe fluidized bed, wherein removing the particles comprises: flowing asecond stream into the chamber through the outlet, wherein flowing thesecond stream acts to create a force at least partially in the samedirection as the centrifugal force field; and collecting the particlespassing through the inlet of the chamber.
 2. The method of claim 1,wherein the particles are cells, the method further comprising:providing the first stream from a cell culture system prior to flowingthe first stream into the chamber, exchanging the media; and deliveringthe cells and exchanged media to the cell culture system after removingthe cells from the fluidized bed.
 3. The method of claim 1, wherein theparticles are cells, wherein manipulating the cells comprisestransfecting the cells, and wherein transfecting the cells comprisescirculating a transfection stream containing a transfection reagentcomplex through the fluidized bed of cells one or more times.
 4. Themethod of claim 1, wherein the particles are cells, wherein manipulatingthe cells comprises electroporating the cells, and whereinelectroporating the cells comprises: applying an electric current to thefluidized bed of cells; and altering the permeability of the cells. 5.The method of claim 4, wherein electroporating the cells furthercomprises: flowing a charged molecule stream containing chargedmolecules into the chamber through the inlet before, concurrently with,and/or after applying the electric current; and incorporating thecharged molecules into the cells.
 6. (canceled)
 7. The method of claim1, wherein manipulating the particles comprises concentrating theparticles, wherein concentrating the particles comprises receiving theparticles in a concentrated particles harvest container after removingthe particles.
 8. The method of claim 1, wherein manipulating theparticles comprises exchanging the media, and wherein exchanging themedia comprises: flowing a new media stream comprising a second mediainto the chamber through the inlet; and replacing at least some of thefirst media in the fluidized bed with the second media.
 9. The method ofclaim 1, wherein manipulating the particles comprises harvesting theparticles, and wherein harvesting the particles comprises receiving theparticles in a particle harvest container after removing the particles.10. The method of claim 1, wherein manipulating the particles comprisesdispensing the particles, and wherein dispensing the particles comprisesreceiving a measured amount of particles in one or more dispensedparticle containers after removing the particles.
 11. The method ofclaim 1, wherein the particles comprise a mixed population of particles,wherein manipulating the particles comprises separating the mixedpopulation of particles, and wherein separating the mixed population ofparticles comprises: removing at least some of the mixed population ofparticles from the fluidized bed; and collecting the at least some ofthe mixed population of particles passing through the outlet of thechamber.
 12. The method of claim 11, wherein removing at least some ofthe mixed population of particles comprises altering the centrifugalforce field and/or the force of the first stream.
 13. The method ofclaim 11, wherein the mixed population of particles are separated bysize, density, and/or shape.
 14. The method of claim 1, whereinmanipulating the particles comprises coating the particles, and whereincoating the particles comprises: flowing a coating stream containing acoating material into the chamber through the inlet; and coating theparticles retained in the fluidized bed with the coating material.
 15. Amethod for separating a mixed population of particles, the methodcomprising: rotating a chamber about a substantially horizontal axis,the chamber having an inlet and an outlet; substantially immobilizing anaffinity matrix in the chamber; flowing a first stream containing afirst media and a mixed population of particles comprising targetparticles and non-target particles into the chamber through the inlet;retaining target particles in the affinity matrix in the chamber; andcollecting the first media and non-target particles passing through theoutlet of the chamber.
 16. The method of claim 15, further comprising:flowing a second stream containing an elution media into the chamberthrough the inlet; releasing the target particles from the affinitymatrix; and collecting the target particles passing through the outletof the chamber. 17-19. (canceled)
 20. A method for fractionatingbiomaterials, the method comprising: rotating a chamber about asubstantially horizontal axis to create a centrifugal force field, thechamber having an inlet and an outlet; flowing a first stream containinga first media and a mixture of biomaterials into the chamber through theinlet, wherein flowing the first stream acts to create a force whichopposes the centrifugal force; selectively precipitating biomaterialsfrom the first stream; forming a fluidized bed of the precipitatedbiomaterials in the chamber, wherein the forces substantially immobilizethe precipitated biomaterials in the fluidized bed by the summation ofvector forces acting on the precipitated biomaterials; then collectingthe first media and the non-precipitated biomaterials passing throughthe outlet of the chamber; and thereafter removing the precipitatedbiomaterials from the fluidized bed, wherein removing the precipitatedbiomaterials comprises: flowing a second stream into the chamber throughthe outlet, wherein flowing the second stream acts to create a force atleast partially in the same direction as the centrifugal force field;and collecting the precipitated biomaterials passing through the inletof the chamber.
 21. The method of claim 20, wherein the biomaterial isprotein. 22-29. (canceled)